Hypoglycemia is the major factor limiting implementation of effective treatment regimens for insulin-dependent diabetes (IDDM). This project continues studies focused on the counterregulatory systems that defend against hypoglycemia, the impact of hypoglycemia of brain fuels and function, and how IDDM and its treatment modify counterregulatory responses to as well as the CNS and peripheral effects of hypoglycemia. The experimental approach relies heavily on the microdialysis technique to directly sample or perturb the local microenvironment of key tissues (e.g., brain, muscle) and the clamp technique to generate a standardized hypoglycemic stimulus. We will determine the role of the ventromedial hypothalamus (VMH) in detecting and triggering responses to hypoglycemia by preventing VMH glucopenia by local glucose perfusion via microdialysis in awake rats, while hypoglycemia is produced in the rest of the body. Changes in VMH neurotransmitter profiles during hypoglycemia will be measured and the hypothesis that the VMH glucoreceptors use similar cellular mechanisms (e.g., GLUT 2, glucokinase, KATP channels) as pancreatic beta cells will be tested. To evaluate the effect of hypoglycemia on human brain metabolism, we will use microdialysis to measure the concentrations of glucose and lactate in brain extracellular fluid (BCF) of patients undergoing intracerebral depth electrode monitoring for intractable epilepsy. Changes in brain ECF fuels will be monitored during hyper- and hypoglycemia and during neuronal activation. The goal is to confirm and extend preliminary data showing a large glucose concentration gradient between blood and brain ECF and an unexpectedly high level of lactate in brain ECF. We will test the hypothesis that antecedent glycemic control modulates the glucose threshold at which brain function becomes impaired during hypoglycemia by performing clamp studies in diabetic BB rats with disparate levels of glycemic control while recording auditory-evoked potentials from electrodes implanted in the inferior colliculus, a brainstem region that is sensitive to hypoglycemia. Mechanisms underlying effects of glycemic control will be examined by measuring ECF glucose, glucose utilization and glucose transporters. Finally, we will use microdialysis and ultrafiltration to simultaneously monitor skeletal muscle and adipose tissue lECF to determine the local concentration of key fuels and catecholamines in peripheral tissues during hypoglycemia and how IDDM and intensive insulin therapy modify them. The aim is to test the hypothesis that changes in intramuscular lipid fuels induced by local sympathoadrenal stimulation play a critical role in glucose counterregulation during insulin-induced hypoglycemia, especially in IDDM. The long-term goal is to generate data that will facilitate the development of new strategies to achieve glycemic control of IDDM more safely.